Introduction

Although light is an indispensable resource (Poorter 2001), both low and high of light availability can limit plant performance (Valladares and Niinemets 2008). While low irradiance in leaves decreases carbon gain and reduces plant growth (Aleric and Kirkman 2005), its excess can cause deleterious effects by inducing photoinhibition (Jordan et al. 2005). However, plants often exhibit a remarkable ability to adjust their physiology, morphology, and anatomy in response to a particular light condition by acclimation, which allows plants to use efficiently the light energy, while avoiding photodamage to their leaves (Givnish 1988; Hanba et al. 2002; Walters 2005; Valladares and Niinemets 2008). The degree of response to increased light varies among species, and may result in altered patterns of growth and biomass allocation (Chazdon et al. 1996; Valladares and Niinemets 2008; Cano et al. 2011).

In response to different light intensities, many plant species are able to acclimate through the development of sun or shade leaves (Givnish 1988; Yano and Terashima 2004). Under low irradiance, plants tend to allocate more resources in leaves than in roots and develop larger and thinner leaves with lower stomatal density and photosynthetic rates (Poorter et al. 2012). In contrast, when exposed to high irradiance, plants have smaller and thicker leaves with higher stomatal density and carbon assimilation rates, investing more resources in root biomass (Givnish 1988; Poorter et al. 2012). Due to these characteristics, sun leaves have higher tolerance to high irradiance and enhanced capacity to dissipate the light excess, whereas shade leaves are prone to suffer the damage caused by excessive light (Demmig-Adams and Adams 1992; Gyimah and Nakao 2007).

The hardening process under high irradiance has been widely used in nurseries as a mechanism for increasing the robustness of seedlings of tropical tree species. Hardy plants are more likely to survive after planting than non-hardened ones and their development can be faster in the field (Jacobs et al. 2005). Studies on morphological, anatomical, and ecophysiological responses of different tropical tree species during the hardening process may contribute to the definition of a hardiness pattern for them. In nurseries that deal with homogenous populations of Pinus and Eucalyptus varieties, relationships between morphological parameters have been used to evaluate the hardiness of the seedling. The stem height by stem base diameter ratio (H/D), the shoot dry mass by root dry mass ratio (SDM/RDM), and the Dickson quality index (DQI), which considers the total dry mass production and its allocation in shoots and roots (SDM/RDM) as well as the H/D (Dickson et al.1960; Claussen 1996) are some examples. However, it is still a challenge to set a hardiness pattern for non-commercial tropical native seedlings based solely on quality indicators, due to the genetic variability of these species.

The knowledge of how plants respond morphologically and physiologically to contrasting light conditions can be useful to explain their occurrence and abundance patterns under specific environmental conditions (Yang et al. 2014), and it is also important for the production of tree seedlings for reforestation of degraded environments (Krause et al. 2001; Oguchi et al. 2006), especially in tropical areas. The understanding of morphological and photosynthetic traits of a desired species when planted in either shade or exposed sites is required for successful reforestation.

This study aimed to contribute to restoration programs by providing information about the high-light acclimation of three light-demanding tree species. For this, hardiness of the seedling was evaluated through the analysis of leaf gas exchange parameters and the morphology and anatomy of seedlings subjected to full sunlight in comparison with those maintained at shaded conditions. The following questions were raised: (1) which photosynthetic adjustments the three species develop in response to different light environments? (2) Which hardiness traits are developed in seedlings of light-demanding tree species during the high-light hardening process in nurseries?

Materials and methods

Study area, species, and experimental design

This research was carried out at the nursery of the “Laboratório de Biodiversidade e Restauração de Ecossistemas” (23°32′44″S, 51°19′83″W) of the Universidade Estadual de Londrina, Londrina, Paraná state, Southern Brazil. Three light-demanding tree species were selected due to their importance in reforestation programs in the region: Heliocarpus popayanensis Kunth (Malvaceae), Guazuma ulmifolia Lam. (Malvaceae), and Aegiphila integrifolia (Jacq.) Moldenke (Lamiaceae). Our purpose was to investigate the performance of seedlings under exactly the same conditions to which they are subjected in the nursery hardening routine. Thus, approximately 1000 seedlings of each species were grown in 50 cm3 polyethylene tubes filled with a mixture of sieved organic compost (cattle manure and litter), carbonized rice husk, and washed sand, enriched with Osmocot® beads, a slow-release fertilizer (15 % N, 10 % P2O5, and 10 % K2O). All plants remained in the growth sector of the nursery until they reached about 20 cm of height. This sector was covered with a polypropylene screen resulting in the incidence of only 40 % of total photosynthetic photon flux density (PPFD). After the growing period, about 40 % of seedlings were maintained in the growth sector (shade; control) and 60 % were transferred to full sunlight at the acclimation sector (sun). The seedlings were kept in these sectors for 168 days. In both sectors, they were watered for 30 min four times per day by an automatic irrigation system.

Leaf gas exchange measurements

Up to 168 days after beginning of the experiment, gas exchange parameters were determined every 14 days on the penultimate fully expanded leaves (without signs of senescence) from nine randomly selected seedlings of each treatment per species. The criterion for leaf choice was always applied even for seedlings of the acclimation sector, regardless the picked leaf has been initially developed under shade or full sun. Therefore, the first measurements were done in the standing leaves, which were developed under shaded conditions. Leaf gas exchange parameters were measured using a Portable Photosynthesis System LI-6400XT (LI-COR Biosciences Inc., Lincoln, NE, USA). A red-blue LED light source attached to the system was used to keep constant the PPFD at 1900 μmol m−2 s−1, enough to saturate the CO2 assimilation rate of the three species (as determined by light response curves). The light-saturated net photosynthetic rate (A max, µmol m−2 s−1), stomatal conductance (g s, mol m−2 s−1), and transpiration rate (E, mmol m−2 s−1) were measured between 8:00 a.m. to 10:00 a.m. The water-use efficiency (WUE, µmol mmol−1) was calculated by the A max/E ratio.

Anatomical analysis

Anatomical characteristics were evaluated at 168 days of the experiment in nine randomly selected seedlings of each treatment per species. We collected segments from the central area of the lamina of the penultimate fully expanded leaf, which were fixed in FAA 70 % (formaldehyde, glacial acetic acid, and ethanol 70 %, 5:5:90) for 72 h, then washed in distilled water, dehydrated in an ethanol-xylol series, and finally embedded in paraffin (Johansen 1940). Transverse 14-µm-thick sections were stained with astrablue—basic fuchsin and mounted in Canadian balsam (Macêdo 1997).

Additional leaf lamina segments (1 cm2) were treated with Jeffrey’s solution (1:1 chromic and nitric acids) for epidermis analyses (Macêdo 1997). Both epidermis faces were stained with astrablue—basic fuchsin and mounted in Canadian balsam (Macêdo 1997).

All slides were deposited at the Laboratório de Anatomia Vegetal, Universidade Estadual de Londrina. Observations and digital photomicrographs of the section were taken under a light microscope (Axioplan Zeiss, Germany). Digital photos were scaled and analyzed with Motic Images Plus 2.0 software (Motic China Group Co. Ltd., Xiamen, China). The thickness (μm) of the lamina, epidermis (adaxial and abaxial face), and mesophyll (palisade and spongy parenchyma) were measured, as well as the stomatal density (the number of stomata mm−2) was calculated.

Morphological measurements

All morphological parameters were recorded in nine seedlings of each treatment per species. Plant height (H-from the base of the plant to the apex) and stem base diameter (D) were recorded at the end of the experiment. Stem base diameter was measured with digital calipers (±0.01 mm) at the soil surface, whereas the stem height was measured with millimeter ruler. We also counted the total number of leaves (TNL) and the number of newly developed leaves during the experiment (NDL).

At the end of the experiment, all leaves of each seedling were scanned in monochromatic format and the readings of the images were obtained by DDA 1.2.0.0 software (Ferreira et al. 2008) for the determination of the total leaf area (LA, cm2) of each seedling. In order to measure the dry masses, the parts of the seedlings were separated (roots, stem, and leaves) and oven-dried at 70 °C to constant mass (approximately 72 h). Then, using a semi-analytical scales, we obtained the root (RDM), stem (StDM), leaves (LDM), shoot (SDM = StDM + LDM), and total (TDM = RDM + SDM) dry masses. Specific leaf area (SLA, cm2 g−1) of each seedling was calculated as the LA to LDM ratio. We also calculated H/D and SDM/RDM ratios and the Dickson quality index, given by DQI = TDM/(H/D + SDM/RDM) (Dickson et al. 1960).

Statistical analysis

Data were tested using one-way analysis of variance (ANOVA) with 5 % significance levels in Statistica 10.0 (Statsoft Inc., Tulsa, USA). The percentage data were transformed (arcsine of the square root of the ratio) to meet the assumption of ANOVA (normal distribution and equal variances).

Results

Irrespective of tree species, no seedling died during the experiment, but during the first weeks the seedlings transferred to the acclimation sector showed some signs of injury, such as yellowing and senescence of mature leaves (data not shown).

In the case of H. popayanensis, full sunlight acclimation induced increases in A max at the beginning (14 and 42 days) and the end (126, 154, and 168 days) of the experiment (Fig. 1a). In comparison with shade seedlings, the sun plants showed lower g s and E during a large extent of the experiment (28, 56 to 126 days, and 56 to 112 days, respectively) (Fig. 1b, c). As a result, higher WUE values were observed in sun seedlings from 56 to 126 days (Fig. 1d). In general, NDL and TNL did not differ between treatments, except at 14 days or at 28, 98, 112, and 126 days, when sun seedlings showed a decrease in NDL (Fig. 1e) or TNL (Fig. 1f), respectively.

Fig. 1
figure 1

Leaf gas exchange and leaf number of Heliocarpus popayanensis grown under 40 % of photosynthetic photon flux density (filled square—shade) or under full sun (open circle—sun) over 168 days of the experiment. a Maximum rate of net photosynthesis (A max), b stomatal conductance (g s), c transpiration rate (E), d water-use efficiency (WUE), e cumulative number of new developed leaves (NDL), and f total number of leaves (TNL), during 56 and 168 days of the experiment, respectively. Asterisk indicates significant differences (P < 0.05) by F test (ANOVA). Verticals bars indicate SE (n = 9)

Full size image

Heliocarpus popayanensis has hypostomatic leaves. Although the stomatal density did not differ between treatments, the thickness of palisade and spongy parenchyma, adaxial epidermis, and leaf lamina was higher in sun plants than in shade ones (Table 1).

Table 1 Leaf anatomic parameters (means ± standard errors, n = 9) of Heliocarpus popayanensis (Hp), Guazuma ulmifolia (Gu), and Aegiphila integrifolia (Ai) seedlings grown under 40 % of photosynthetic photon flux density (shade) or full sunlight (sun) after 168 days of experiment
Full size table

Sun seedlings of H. popayanensis showed lower H, but D did not differ in comparison with shade seedlings (Table 2). LA, SLA, and LDM were reduced during acclimation (Table 2). DQI was higher, while H/D and SDM/RDM were lower in sun seedlings (Table 2), indicating higher hardiness when compared to shade seedlings.

Table 2 Growth parameters (mean ± standard errors, n = 9) of Heliocarpus popayanensis (Hp), Guazuma ulmifolia (Gu), and Aegiphila integrifolia (Ai) seedlings, grown under 40 % of photosynthetic photon flux density (shade) or under full sun (sun) after 168 days of the experiment
Full size table

At initial phase of the experiment (up to 70 days), the hardening process reduced the A max of G. ulmifolia seedlings (Fig. 2a). Additionally, lower g s and E were detected in sun seedlings compared to shade ones until 42 days of the experiment (Figs. 2b, c). However, at the second part of the experiment, there was a recovery in the A max of the sun seedlings (Fig. 2a), as well as they showed higher values of g s and E in comparison with shade plants (Figs. 2b, c).

Fig. 2
figure 2

Leaf gas exchange and leaf number of Guazuma ulmifolia grown under 40 % of photosynthetic photon flux density (filled square—shade) or under full sun (open circle—sun) over 168 days of the experiment. a Maximum rate of net photosynthesis (A max), b stomatal conductance (g s), c transpiration rate (E), d water-use efficiency (WUE), e cumulative number of new developed leaves (NDL) and f total number of leaves (TNL), during 56 and 168 days of the experiment, respectively. Asterisk indicates significant differences (P < 0.05) by F test (ANOVA). Verticals bars indicate SE (n = 9)

Full size image

In general, WUE was lower in sun plants, especially at the end of the experiment (Fig. 2d). A TNL reduction was observed in sun seedlings from 28 days (Fig. 2f), while NDL reduction was observed only at 56 days in relation to shade seedlings (Fig. 2e).

Anatomical analysis showed that G. ulmifolia present hypostomatic leaves. The alterations induced by high-light acclimation were increases in stomatal density and palisade parenchyma thickness (Table 1).

The dry masses of most organs were lower in sun seedlings (only RDM remained unchanged) (Table 2). H and SLA did not differ between treatments, but D and LA were lower in sun seedlings (Table 2). In these plants, the H/D was higher, while SDM/RDM and DQI were lower when compared to shade seedlings (Table 2).

Sun seedlings of A. integrifolia showed A max reduction at 14 and 28 days when compared to shade seedlings (Fig. 3a). However, after this initial phase, A max of sun plants increased to values similar or even higher than the shaded control (Fig. 3a). High-light acclimation led to an increase in E (Fig. 3c), which was followed by higher g s in some days (from 56 to 98 days, and 126 days) (Fig. 3b). In sun seedlings, WUE reduction was observed in comparison with shade seedlings, except from 140 and 168 days (Fig. 3d). In general, NDL and TNL did not differ between treatments (Fig. 3e, f). However, it should be noted that the species shows a deep decrease in the total leaf number during experiment in both treatments.

Fig. 3
figure 3

Leaf gas exchange and leaf number of Aegiphila integrifolia grown under 40 % of photosynthetic photon flux density (filled square—shade) or under full sun (open circle—sun) over 168 days of the experiment. a Maximum rate of net photosynthesis (A max), b stomatal conductance (g s), c transpiration rate (E), d water-use efficiency (WUE), e cumulative number of new developed leaves (NDL), and f total number of leaves (TNL), during 56 and 168 days of the experiment, respectively. Asterisk indicates significant differences (P < 0.05) by F test (ANOVA). Verticals bars indicate standard errors (n = 9)

Full size image

Aegiphila integrifolia also showed hypostomatic leaves. In sun seedlings, the stomatal density was higher and the abaxial epidermis, palisade and spongy parenchyma, and leaf lamina were thicker than shade seedlings (Table 1).

We did not observe differences in H and D between treatments, but the dry masses, LA, and SLA were lower in sun seedlings of A. integrifolia (Table 2). The H/D and SDM/RDM did not differ between treatments; however, the DQI was lower in sun plants (Table 2).

Discussion

Our study provided a characterization of the physiological, anatomical, and morphological changes induced by the full sunlight exposition of seedlings, in a nursery, of three light-demanding tree species used in reforestation programs in Brazil. The seedlings of H. popayanensis, G. ulmifolia, and A. integrifolia showed different responses to full sun exposition, showing species-specific acclimation in the evaluated parameters. However, all of them developed characteristics of hardiness, which may favor their initial establishment at the field.

The increase in light intensity after the transfer of seedlings to acclimation sector did not stimulate the development of new leaves in any species. However, G. ulmifolia and H. popayanensis seedlings showed a reduction in TNL after the transfer, due to the shedding of older leaves, which indicated faster leaf senescence in sun plants. Indeed, sun-grown plants usually exhibit faster leaf senescence, but they also show higher leaf production than shade plants of the same species (Chabot and Hicks 1982; Sims and Pearcy 1992), a pattern that was not observed in this study. This may have occurred because in high-light environments, there is usually higher heat loads and vapor pressure deficits (Sessa and Givnish 2014), and seedlings possibly underwent drought stress in the acclimation sector, especially in warmer days, due to the low capacity of the tubes to retain water. Water shortage can induce leaf shedding, which should attenuate water stress and reduce the risk of hydraulic failure of plants (Cortina et al. 2013). At the end of the experiment, all the A. integrifolia seedlings were healthy. The decrease of leaf number in both treatments must have been caused by some factor inherent to the developmental process of the seedlings. Therefore, the progressive loss of leaves was not caused by contrasting abiotic conditions between treatments.

Leaf gas exchange analysis demonstrated an initial decrease in A max of G. ulmifolia and A. integrifolia seedlings after the transfer to full sunlight. This depression in CO2 assimilation rate was probably a consequence of the inability of mature leaves expanded under shade to dissipate excess excitation energy and develop effective photoprotective mechanisms, thus resulting in photoinhibition, as previously proposed for other tree species (Claussen 1996; Kitao et al. 2000; Matsuki et al. 2003; Endres et al. 2010; Gonçalves et al. 2013). The effects of photoinhibition can be minimized by the production of new leaves with biochemical and structural traits that lead to acclimation to high irradiances (Mulkey and Pearcy 1992; Kitao et al. 2000; Yamashita et al. 2000; Cuzzol and Milanez 2012). Thus, the recovery of A max of G. ulmifolia and A. integrifolia seedlings observed after the initial phase of the experiment might be a consequence of the production of sun-acclimated leaves. Similarly, the observation that, in general, H. popayanensis photosynthetic rates were not negatively affected by high irradiances may be related with faster rates of development of new leaves of this species.

Indeed, in the three studied species, the leaves expanded under full sunlight presented some of the morphoanatomical traits typical of sun leaves previously described in the literature (Boardman 1977; Givnish 1988; Ivancich et al. 2012), such as lower LA and SLA, thicker palisade and spongy parenchyma, and higher leaf thickness and stomatal density. In particular, the thickening of palisade parenchyma was observed in acclimated leaves of all species, which may represent a structural mechanism for increasing photosynthetic rates under high irradiances (Boardman 1977; Oguchi et al. 2005; Ivancich et al. 2012). The higher development of chlorenchyma is usually associated with a reduction of SLA (Poorter 1999; Hanba et al. 2002; Ivancich et al. 2012), a pattern that was observed in H. popayanensis and A. integrifolia. In these species, the higher SLA of shade seedlings may contribute to the increase of light absorption in environments of low irradiance (Poorter 1999). However, due to the greater surface area per leaf volume, this characteristic may decrease the control of water loss (Ivancich et al. 2012), thus augmenting the probability of dehydration after the transfer to the field.

In addition to the influence on SLA, the thickening of chlorenchyma induced by high irradiance stimulates the production of chloroplasts, what may result in an increase of CO2 assimilation per leaf area (Evans and Poorter 2001; Terashima et al. 2001). This might explain the higher A max values of H. popayanensis and A. integrifolia sun seedlings in comparison with shaded ones during the experiment, as suggested for other tree species (Martínez-Pastur et al. 2007; Ivancich et al. 2012), as well as the recovery of A max values of G. ulmifolia and A. integrifolia sun seedlings. This ability of seedlings to develop physiological adjustments to high irradiances has been associated with higher probability of survival in the field (Gyimah and Nakao 2007).

The increase in photosynthetic rate at high light may occur at expenses of higher stomatal conductance, which causes greater water loss by transpiration (Lee et al. 2000; Hanba et al. 2002). In fact, the increase of E resulted in a WUE reduction in G. ulmifolia and A. integrifolia sun plants, as verified by Matsuki et al. (2003) and Endres et al. (2010) in the species studied by them. Nevertheless, the increased water loss by transpiration has been associated with a reduction of leaf temperature, therefore minimizing the risks of overheating and photoinhibition in plants under high light intensities (Krause et al. 2001).

Differently, together with higher A max values, sun seedlings of H. popayanensis showed lower g s in most days of analysis, which resulted in a reduction of E and an increase of WUE compared to shade seedlings. It suggests that this species was able to assimilate carbon with a lower water loss, as verified in Acer rufinerve Sieb. et Zucc. plants growing under high irradiance (Hanba et al. 2002). The reduction in g s in H. popayanensis sun seedlings reinforces the possibility that plants have suffered water shortage in some periods in the acclimation sector. Stape et al. (2001) reported that in seedlings grown in the same tubes used here (50 cm3), rapid root growth can cause restrictions in water drainage of tubes, and then decrease leaf water potential and increase stomatal resistance. Anyway, low leaf g s in drier sites can protect the root-to-leaf hydraulic pathway from cavitation (Brodribb and Holbrook 2006), reduce plant transpiration rate, and favor seedling survival in water-stressed conditions after planting (Delpérée et al. 2003).

It is also noteworthy that high-light acclimation did not affect the total biomass of H. popayanensis seedlings, whereas the biomass of G. ulmifolia and A. integrifolia sun seedlings decreased when compared to control ones. The decrease in total dry mass in sun seedlings of these species may be a consequence of reduced CO2 assimilation rates (per leaf area) observed at the first part of the experiment, together with the reduced leaf area at the end of the experiment (which decreases plant total photosynthesis). Similarly, the excess of light energy has been associated with photoinhibition and limited biomass production in different tree species (Long et al. 1994; Claussen 1996; Kitao et al. 2000; Gonçalves et al. 2005).

Despite the decrease in total dry mass, the hardening process did not affect root dry mass of G. ulmifolia seedlings, leading to decreased SDM/RDM ratio, a result that was also observed for H. popayanensis sun seedlings. In seedlings of Eucalyptus sp., the decrease in SDM/RDM ratio was attributed to the application of drought cycles in nursery, due to its negative effect on leaf expansion, but not on root development (Rhizopoulou and Davies 1993; Stape et al. 2001). A lower SDM/RDM ratio implies that biomass was preferentially allocated in roots in comparison with aerial organs. In plants growing under high irradiances, this response favors water and nutrient absorption in detriment to water loss by transpiration (Poorter 2001; Mielke and Schaffer 2010), which would contribute to initial growth of the plants after their transfer to the field (Claussen 1996; Moraes et al. 2010; Freitas et al. 2012).

Despite not presenting the thickening of stem base diameter, H. popayanensis sun plants presented reduced stem height, resulting in lower H/D ratio in comparison with shade seedlings. It suggests that these seedlings showed a greater equilibrium in the development of the different parts and that the stem base diameter was compatible with the height, what might imply in seedlings that would be more tolerant to the conditions found at the field (Campos and Uchida 2002). Sun seedlings of H. popayanensis also presented a higher DQI value when compared to shade seedlings. Taken together with the lower H/D and SDM/RDM ratios, it indicates that analysis of these morphological parameters was adequate for expressing the quality of H. popayanensis sun seedlings, suggesting higher hardiness at the end of the experiment. However, it was not valid for G. ulmifolia and A. integrifolia seedlings.

In G. ulmifolia, sun seedlings presented higher H/D, whereas the hardening process did not affect this ratio in A. integrifolia (as well as the SDM/RDM). Moreover, the DQI was lower in sun seedlings of G. ulmifolia and A. integrifolia in comparison with shaded ones. These results do not necessarily indicate low quality of the seedlings, given that there are not in the literature reference values for the seedlings of different tropical tree species (Leles et al. 2006; Ferraz and Engel 2011). However, Fonseca et al. (2002) recorded higher DQI in seedlings of Trema micrantha (L.) Blume grown under full sunlight. Thus, the quality indexes are not necessarily adequate for expressing the hardiness of all tropical tree seedlings, especially if we consider that they constitute more genetically heterogeneous populations. More studies are necessary for the proposal of quality indexes more adequate for nurseries that deal with different tropical tree species.

More importantly, irrespective of the quality indexes, the seedlings of the three species analyzed in the present study developed traits of hardiness after acclimation under full sunlight. Sun plants of H. popayanensis presented physiological (increases in A max and WUE) and morphoanatomical responses (thicker mesophyll and palisade parenchyma), in addition to the reduction of H/D and SDM/RDM, and higher DQI. In A. integrifolia seedlings subjected to high irradiances, leaves also developed morphoanatomical traits of hardiness (thicker mesophyll and palisade parenchyma and higher stomatal density) and presented higher A max values at the end of the experiment (in spite of the initial drop in this parameter). In G. ulmifolia, at the end of the hardening process, although A max did not increase compared to shade seedlings, the leaves developed under full sunlight did not present signs of photoinhibition and exhibited structural characteristics of hardiness, such as thickening of palisade parenchyma, higher stomatal density, and lower LA and SDM/RDM. Overall, these characteristics induced by the hardening process in each species might allow better conditions of growth and survival for these seedlings in the field.